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Molecular integration of casanova in the Nodal signalling pathway controlling endoderm formation

Tazu O. Aoki^1,*, Nicolas B. David^1,*, Gabriella Minchiotti², Laure Saint-Etienne¹, Thomas Dickmeis³, Graziella M. Persico², Uwe Strähle³, Philippe Mourrain¹ and Frédéric M. Rosa^1,

¹ U 368 INSERM, Ecole Normale Supérieure, 46, rue d’Ulm, F-75230 Paris Cedex 05, France
² International Institute of Genetics and Biophysics, CNR, Via G. Marconi, 12-80125 Naples, Italy
³ Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, BP 163, F-67404 Illkirch Cedex, CU de Strasbourg, France
^* These two authors contributed equally to this work

View larger version (64K): [in a new window]   Fig. 1. Dynamics of expression of bon/mixer, fau/gata5, casanova and sox17 genes in wild-type embryos at 40% epiboly (A-H), 50% epiboly (I-L), shield (M-P) and 70-80% epiboly (Q-T) stages. (A-D) Animal pole views; (E-T) lateral views, dorsal to the right. At 40% epiboly, whereas bon/mixer (A,E) and gata5/fau (B,F) are homogeneously expressed in large marginal domains, casanova expression is mosaic and preferentially restricted to the most marginal blastomeres of the dorsal side (C,G). At this stage, sox17 is expressed only in the superficial and marginal cells of the dorsal side (D,H). At 50% epiboly, expression patterns of bon/mixer (I), gata5/fau (J) and sox17 (L) are roughly unchanged. The casanova pattern is still mosaic but it is found throughout the margin and in the forerunner cells (K). At the shield stage, bon/mixer (M) and gata5/fau (N) are expressed in more germ ring blastomeres. Cells expressing casanova have begun to involute and abut the YSL, except the forerunner cells, which remain superficial (O). The sox17 gene is expressed in deep cells abutting the YSL in the dorsal axis (P). After the onset of gastrulation, bon/mixer (Q) is no longer expressed, whereas fau/gata5 (R), casanova (S) and sox17 (T) are expressed in the scattered endodermal cells (arrowhead); casanova (S) and sox17 (T) are still expressed in the forerunner cells. (U,V) Close up of the mosaic pattern of casanova at the dorsal margin (U) and lateral margin (V) of embryos at 40% epiboly stage (notice the isolated blastodermal cas-positive cells (arrowheads); the dotted lines mark the YSL-blastoderm frontier). (W-Y) Cross sections of embryos following whole-mount in situ hybridization with casanova at 50% epiboly (W), shield (X) and 70-80% epiboly stage (Y), animal pole up (arrows point to YSL nuclei). The cas positive cells involute at the margin, abut YSL and spread over the whole embryos with a scattered pattern.

View larger version (85K): [in a new window]   Fig. 2. Expression of casanova requires Nodal signalling. (A-D) Expression of casanova requires zygotic contribution of oep. (A,B) Animal pole views, dorsal to the right. (C,D) Dorsal views. Compared with wild-type controls (A,C), casanova endodermal expression is not initiated or maintained in blastula (B, 40% epiboly) nor during gastrulation (D, 70-80% epiboly) in Zoep homozygous mutants. Expression in the forerunner cells and YSL is not affected. (E,F) Induction of cas upon Nodal signalling activation is cell autonomous. In late blastula, a few wild-type cells expressing nls-lacZ alone or combined with tar* were transplanted to the animal pole of a host wild-type embryo. During gastrulation (60% epiboly) tar* expressing cells transplanted to the animal pole (brown nuclear staining) autonomously express casanova (F; 96%, n=93) whereas control cells do not (E; 100%, n=12).

View larger version (82K): [in a new window]   Fig. 3. Rescue of endodermal markers in MZoep by Cripto protein injection at different times. (A) Experimental procedure. Synchronized MZoep embryos were injected with Cripto protein together with rhodamine dextran as a tracer at appropriate stages (from high to 40% epiboly). Subsequently, embryos exhibiting homogeneous rhodamine fluorescence were sorted, fixed and analysed by in situ hybridization (B-S). (B-N) Lateral (C-E,G-I,N) or dorsal views. At 80% epiboly, four different classes of expression pattern of cas (B-E), sox17 (F-I) and foxa2 (J-N) can be identified in Cripto-injected MZoep embryos. When Cripto was injected at 40% epiboly, most embryos do not express foxa2 in endodermal cells but still express it in axial mesoderm (M). (O-S) Staining of fkd7 in Cripto-injected MZoep embryos at 30 hpf. Anterior to the left. Four classes were also defined. (O) Class I exhibits almost normal expression of fkd7 in the pharynx (white arrow) and in the gut (black arrow). (P) Class II exhibits incomplete expression of fkd7, often lacking the pharyngeal endoderm (white arrow) but have an almost normal expression in the gut (black arrow). Class III has only a few fkd7 positive cells (Q, arrow). Class IV shows no endodermal fkd7 staining (R,S).

View larger version (21K): [in a new window]   Fig. 4. Frequency of endodermal markers rescue in MZoep by Cripto-injection at different times, displaying the frequencies corresponding to the four classes described in Fig. 3. Full rescue (Class I) was predominantly observed until sphere stage. Reduction of the number of endodermal cells expressing cas, sox17 or foxa2 was observed when Cripto protein was injected at dome (Classes II and III). At 40% epiboly, Cripto protein was not able to induce these markers in almost all injected embryos (Class IV). Embryos probed for foxa2 show that axial mesoderm but not endoderm was rescued when Cripto protein was injected at 40% epiboly (Fig. 3M).

View larger version (46K): [in a new window]   Fig. 5. Expression of bon/mixer is required for casanova expression. (A,B) Dorsal views, 70-80% epiboly. Compared with the control (A), casanova is normally expressed in most faust mutant embryos, but is sometimes slightly reduced (B). (C,D) Lateral views, 40% epiboly. (E,F) Dorsal views, tail bud. Compared with the control (C,E), there are many fewer cas positive cells in bon mutant embryos from late blastula (D) to late gastrula (F). (G-J) Dorsal views, 70-80% epiboly. Compared with the control (G,I), Xmixer overexpression increases the number of cells expressing casanova in wild-type embryos (H) but not in MZoep mutants (J). (K,L) Combination of Xmixer and fau/gata5 (L, compare with control K) induces, in a few embryos (5%, n=20), a very small number of cas positive cells, which never reach the YSL.

View larger version (105K): [in a new window]   Fig. 6. Expression of cas represses mesodermal markers, induces endodermal markers and can change the fate of cells to an endodermal identity. (A-F) Anterior to the left, dorsal to the top. (A,D) Clonal progeny of one marginal blastomere injected with gfp alone (A) or combined with cas (D) RNA at the 16-cell stage. At 24 hpf, expression of cas leads to a more frequent colonization of endodermal territories, like pharynx (arrowhead in D). (B-C, E-F) This ability to change the cell fate was confirmed by transplant experiments. By blastula stage, a few cells expressing only the gfp gene (B-C) or both gfp and cas (E-F) were transplanted to the margin of host embryos. At 24 hpf, cells expressing cas more frequently became involved in endodermal derivatives such as pharynx (E) and gut (arrowhead in F). Dorsal (G-H, O-P) and animal pole (I-L, Q-R) views of shield and 75% epiboly embryos. (G-L) Compared with the lacZ injected siblings (G,I,K), injection of cas RNA at the 8/16-cell stage induces expression of endodermal markers sox17 (H; 100%, n=23), foxa2 (J; 92%, n=12) and gata5 (L; 93%, n=15). (M,N) This induction is cell autonomous, as revealed by transplant experiments (the nuclei of grafted cells has been stained in brown by immunodetection of nuclear ß-galactosidase, used as a lineage tracer). By contrast, mesodermal markers ntl (O,P) and tbx6 (Q,R) are repressed by overexpression of cas (arrowheads in P and R; 86%, n=21 and 78%, n=18, respectively). Conversely, overexpression of ntl represses cas expression at the margin (arrowhead in T, compare with S).

View larger version (67K): [in a new window]   Fig. 7. Expression of casanova rescues late differentiation of endoderm and early endodermal marker expression in oep and bon mutants. Compared with 24 hpf MZoep (A), Zoep (B) and bon (C) controls, which are deficient in endodermal structures and in the endodermal differentiation marker fkd7, casanova overexpression can sometimes restore the differentiation of pharynx in MZoep mutants (D) (11%, n=97) and efficiently restores differentiation of pharynx and gut in Zoep (E,F) (88%, n=42) and bon mutants (G) (100%, n=17), as evidenced by fkd7 expression (arrowheads). Overexpression of casanova also increases the number of cells expressing the early endodermal markers sox17 and foxa2 in MZoep (H-K) and bon (L-O) embryos.

View larger version (56K): [in a new window]   Fig. 8. Functional Nodal signalling is required cell autonomously to allow Cas to change the cell fate. (A) Experimental procedure. MZoep or wild-type embryos were injected with a lineage tracer (gfp for MZoep embryos and rhodamine for wild-type embryos) alone or combined with cas RNA. By the blastula (sphere-dome stage), one mutant cell and one wild-type cell were transplanted to the margin of a wild-type host (B). During gastrulation, both mutant and wild-type cells, expressing cas or not, involute and remain close to or in contact with the YSL (C,D). The dotted line indicates the position of the margin. (E) Anterior to the left, dorsal to the top. At 24 hpf, MZoep cells hardly ever took part in endodermal derivatives (only six intestinal cells in one embryo, out of 24 embryos), whereas wild-type cells expressing cas frequently participated in the endoderm (58%, n=31; Fig. 6). Abbreviations: s, somites; y, yolk.

View larger version (8K): [in a new window]   Fig. 9. A model for endoderm formation and differentiation, adapted from Alexander and Stainier (Alexander and Stainier, 1999). Our work suggests the involvement of an unknown factor (X) acting with bon/mixer and fau/gata5 for cas induction, and it also shows the requirement for an independent Y pathway induced by Nodal signalling to allow proper endoderm differentiation.